Efforts towards an effective HIV vaccine have been greatly hampered by the high antigenic variability of the HIV envelope spike (Env). Despite this variability, an increasing number of monoclonal antibodies (mAbs) that are capable of recognizing a diversity of cross-clade isolates, termed broadly neutralizing antibodies (bnAbs), have been isolated from chronically infected donors (Walker, L. M. et al. Science 326, 285-289 (2009); Doria-Rose, N. A. et al. Nature (2014) doi:10.1038/nature13036; Kwong, P. D. & Mascola, J. R. Immunity 37, 412-425 (2012); Walker, L. M. et al. Nature 477, 466-470 (2011); Bonsignori, M. et al. J Virol 85, 9998-10009 (2011); Burton, D. R. et al. Cell Host Microbe 12, 396-407 (2012); Wu, X. et al. Science 329, 856-861 (2010); Huang, J. et al. Nature 491, 406-412 (2012); Scheid, J. F. et al. Science 333, 1633-1637 (2011); Mouquet, H. et al. Proc Natl Acad Sci USA 109, E3268-77 (2012); Klein, F. et al. J Exp Med 209, 1469-1479 (2012); Falkowska, E. et al. Immunity (2014) doi:10.1016/j.immuni.2014.04.009; Blattner, C. et al. Immunity (2014) doi:10.1016/j.immuni.2014.04.008). However, even for a family of bnAbs targeting the same epitope region, substantial differences in potency and breadth are found, which likely reflect fine details in epitope recognition. The isolation of families of bnAbs with different neutralization profiles against each site then serves as powerful tools to decipher the epitope features that are most important for neutralization breadth and potency.
Multiple methods have been developed to isolate bnAbs. Hybridoma and phage display techniques were used to isolate the first generation of bnAbs including 2F5, b12, 2G12, 4E10 and Z13 (Burton, D. R. et al. Science 266, 1024-1027 (1994); Muster, T. et al. J Virol 68, 4031-4034 (1994); Burton, D. R. et al. Proc Natl Acad Sci USA 88, 10134-10137 (1991); Zwick, M. B. et al. J Virol 75, 10892-10905 (2001); Barbas, C. F. et al. Proc Natl Acad Sci USA 89, 9339-9343 (1992); Trkola, A. et al. J Virol 70, 1100-1108 (1996); Conley, A. J. et al. Proc Natl Acad Sci USA 91, 3348-3352 (1994); Buchacher, A. et al. AIDS Res Hum Retroviruses 10, 359-369 (1994)). These antibodies exhibit a range of neutralization breadth against primary isolates from 30-90%, but have moderate neutralization potency (median IC50 of ˜2-4 μg/mL), suggesting that the elicitation of relatively high serum titers would be required to afford sterilizing immunity in vivo, given the observed relationship between neutralization in vitro and protection in vivo (Parren, P. W. et al. J Virol 75, 8340-8347 (2001); Hessell, A. J. et al. PLoS Pathog 5, e1000433 (2009); Mascola, J. R. et al. J Virol 73, 4009-4018 (1999)). Access to infected donors who have high serum titers of bnAbs (Simek, M. D. et al. J Virol 83, 7337-7348 (2009); Gray, E. S. et al. J Virol 85, 4828-4840 (2011)) and the availability of newer approaches for isolating human mAbs have recently enabled the discovery of a new generation of more potent bnAbs (Walker, L. M. et al. Science 326, 285-289 (2009); Doria-Rose, N. A. et al. Nature (2014) doi:10.1038/nature13036; Walker, L. M. et al. Nature 477, 466-470 (2011); Bonsignori, M. et al. J Virol 85, 9998-10009 (2011); Wu, X. et al. Science 329, 856-861 (2010); Huang, J. et al. Nature 491, 406-412 (2012); Scheid, J. F. et al. Science 333, 1633-1637 (2011)).
The first of these newer approaches involves the sorting and activation of large numbers of memory B cells using cytokine-secreting feeder cells and the subsequent high throughput screening of supernatants for neutralization. This method led to the identification and characterization of the first of this new generation of bnAbs, PG9 and PG16 (Walker, L. M. et al. Science 326, 285-289 (2009)), and has since revealed several new sites of vulnerability to bnAb recognition on Env (Walker, L. M. et al. Science 326, 285-289 (2009); Doria-Rose, N. A. et al. Nature (2014) doi:10.1038/nature13036; Walker, L. M. et al. Nature 477, 466-470 (2011); Bonsignori, M. et al. J Virol 85, 9998-10009 (2011); Wu, X. et al. Science 329, 856-861 (2010); Huang, J. et al. Nature 491, 406-412 (2012)). One of these sites, targeted by the bnAbs PG9/PG16, PGT141-145, CH01-04, and CAP256-VRC26.01-12, is situated at the trimer apex and is centered around the glycan at position 160 on Env (Walker, L. M. et al. Science 326, 285-289 (2009); Doria-Rose, N. A. et al. Nature (2014) doi:10.1038/nature13036; Walker, L. M. et al. Nature 477, 466-470 (2011); Bonsignori, M. et al. J Virol 85, 9998-10009 (2011); Julien, J.-P. et al. Proc Natl Acad Sci USA 110, 4351-4356 (2013)). Indeed, recent structural analyses have revealed that these bnAbs bind to a conserved β-sheet structure located at the trimer apex, mainly in the V2 region, but also spanning V1 and V3 (Julien, J.-P. et al. Proc Natl Acad Sci USA 110, 4351-4356 (2013); Julien, J.-P. et al. Science (2013) doi:10.1126/science.1245625; McLellan, J. S. et al. Nature 480, 336-343 (2011); Pancera, M. et al. Nat Struct Mol Biol 20, 804-813 (2013)). In light of these new structural findings, Applicants henceforth refer to the V2-glycan bnAbs as trimer-apex glycan-dependent bnAbs. Using this screening method, bnAbs targeting other sites of vulnerability on Env were found, including the PGT121-137 antibodies, which target the high-mannose patch epitope centered around the glycan at position N332 (Walker, L. M. et al. Nature 477, 466-470 (2011); Mouquet, H. et al. Proc Natl Acad Sci USA 109, E3268-77 (2012); Julien, J.-P. et al. Science (2013) doi:10.1126/science.1245625; Pejchal, R. et al. Science 334, 1097-1103 (2011); Kong, L. et al. Nat Struct Mol Biol (2013) doi:10.1038/nsmb.2594; Sok, D. et al. PLoS Pathog 9, e1003754 (2013)), the antibody 10E8, which targets the MPER site (Huang, J. et al. Nature 491, 406-412 (2012)), and finally the PGT151-158 antibodies, which target a region at the interface of gp41 and gp120 (Falkowska, E. et al. Immunity (2014) doi:10.1016/j.immuni.2014.04.009; Blattner, C. et al. Immunity (2014) doi:10.1016/j.immuni.2014.04.008).
An alternative method for bnAb isolation involves the use of soluble Env molecules or scaffold proteins as baits to select single IgG+ memory B cells of interest by cell sorting (Wardemann, H. et al. Science 301, 1374-1377 (2003); Scheid, J. F. et al. Nature 458, 636-640 (2009)). This method has cost advantages and does not demand the automated procedures associated with direct neutralization screening. Indeed, this antigen selection method successfully isolated a number of bnAbs from various donors against the CD4bs and the supersite surrounding the N332 glycan (Wu, X. et al. Science 329, 856-861 (2010); Scheid, J. F. et al. Science 333, 1633-1637 (2011); Mouquet, H. et al. Proc Natl Acad Sci USA 109, E3268-77 (2012)). Soluble baits have not, however, been successful at isolating antibody responses targeting quaternary epitopes, including the site surrounding the N160 glycan, as the protein constructs used to date have not properly mimicked native Env trimers. To address this problem, GFP-labeled 293T cells that express cell surface Env for sorting, called GFP-293TBaL, have been used recently to isolate antibodies 3BC176 and 3BC315 (Klein, F. et al. J Exp Med 209, 1469-1479 (2012); Gaebler, C. et al. J Immunol Methods 397, 47-54 (2013)). These antibodies do not bind soluble monomeric gp120 but do bind Env trimer, demonstrating utility of the approach, but the method was described to be inefficient compared to the use of soluble protein baits (Klein, F. et al. J Exp Med 209, 1469-1479 (2012); Gaebler, C. et al. J Immunol Methods 397, 47-54 (2013)).
The recently developed soluble BG505 SOSIP.664 gp140 trimer is a largely faithful antigenic mimic of native Env, as indicated by the strong binding of multiple bnAbs, including to quaternary-structure epitopes, and the very weak or absent binding of non-neutralizing antibodies (Sanders, R. W. et al. PLoS Pathog 9, e1003618 (2013)). The favorable antigenic profile of these trimers opens the possibility of their use for isolating quaternary-specific antibodies by single-cell sorting.
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